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1 Article An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF- B and MAPK pathways Yu-Li Lin,*, Yu-Chih Liang, Yu-Shan Tseng, Hsin-Yi Huang, Shu-Yu Chou, Ruey-Shyang Hseu, Ching-Tsan Huang, and Bor-Luen Chiang*,,1 *Department of Medical Research, Graduate Institute of Clinical Medicine, College of Medicine, and Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan, Republic of China; and Graduate Institute of Biomedical Technology, College of Medicine, Taipei Medical University, Taipei, Taiwan, Republic of China RECEIVED JULY 25, 2008; REVISED MAY 1, 2009; ACCEPTED MAY 12, DOI: /jlb ABSTRACT Ganoderma lucidum, an oriental medicinal mushroom, has been widely used in Asia to promote health and longevity. LZ-8 is a protein derived from the fungus G. lucidum and has immunomodulatory capacities. In this study, we investigated the immune modulatory effects of rlz-8 on human monocyte-derived DCs. Treatment of DC with rlz-8 resulted in the enhanced cell-surface expression of CD80, CD86, CD83, and HLA-DR, as well as the enhanced production of IL-12 p40, IL-10, and IL- 23, and the capacity for endocytosis was suppressed in DCs. In addition, treatment of DCs with rlz-8 resulted in an enhanced, naïve T cell-stimulatory capacity and increased, naïve T cell secretion of IFN- and IL-10. Neutralization with antibodies against TLR4 inhibited the rlz-8-induced production of IL-12 p40 and IL-10 in DCs. rlz-8 can stimulate TLR4 or TLR4/MD2-transfected HEK293 cells to produce IL-8. These results suggested an important role for TLR4 in signaling DCs upon incubation with rlz-8. Further study showed that rlz-8 was able to augment IKK, NF- B activity, and also I B and MAPK phosphorylation. Further, inhibition of NF- B by helenalin prevented the effects of rlz-8 in the expression of CD80, CD86, CD83, and HLA-DR and production of IL-12 p40 and IL-10 in various degrees. To confirm the in vitro data, we investigated the effect of rlz-8 further on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with OVA/ rlz-8 showed that the anti-ova IgG2a, IFN-, and IL-2 were increased significantly compared with OVA alone Abbreviations: BMGY buffered glycerol-complex medium, BMMY buffered methanol-complex medium, crpmi complete RPMI, DC dendritic cell, EAE experimental allergic encephalomyelitis, EU ELISA unit(s), h human, HEK human embryo kidney, IKK I B kinase, MD2 myeloid differentiation protein 2, MFI mean fluorescence intensity, NP-40 Nonidet P-40, P phosphorylated, PS-G polysaccharide from G. lucidum, rlz-8 recombined Ling Zhi-8 in BALB/c mice. In conclusion, our experiments demonstrated that rlz-8 can effectively promote the activation and maturation of immature DCs, preferring a Th1 response, suggesting that rlz-8 may possess a potential effect in regulating immune responses. J. Leukoc. Biol. 86: ; Introduction Ganoderma lucidum, a popular medicinal mushroom, has been widely used in traditional Chinese medicine in many Asian countries during the past two millennia. G. lucidum has been reported to be effective in modulating immune functions and inhibiting tumor growth and allergic disease [1] and in the treatment of chronic hepatopathy, hypertension, and hyperglycemia [2]. The PS-G is a branched (1 6)- -D-glucan moiety. Studies have demonstrated the antineoplastic action of G. lucidum and attributed it to the activated host immune response [3, 4]. G. lucidum polysaccharide and triterperoid were regarded as the major bioactive substances until a new family of fungal immunomodulatory protein, LZ-8, was isolated and purified from the mycelia of G. lucidum in 1989 [5, 6]. The native form of LZ-8 is a noncovalently linked homodimer with an apparent molecular weight of 24 kda. Each polypeptide consists of 110 aa residues with an acetylated N terminus and a molecular mass of 12 kda. In this study, LZ-8 exerted its hemagglutination activity on SRBCs. No aggregation was observed between human RBCs in the presence of LZ-8, and it could function as a potent suppressor of BSA-induced anaphylaxis in CFW mice in vitro. Moreover, mitogenic activity has also been reported [7]. DCs are one of the major professional APCs, whose primary function is to capture, process, and present antigens to 1. Correspondence: Department of Pediatrics, National Taiwan University Hospital, No. 7, Chungshan South Road, Taipei, Taiwan, R.O.C /09/ Society for Leukocyte Biology Volume 86, October 2009 Journal of Leukocyte Biology 877

2 unprimed T cells [8, 9]. Immature DCs reside in nonlymphoid tissues, where they can capture and process antigens. Thereafter, DCs migrate to the T cell areas of lymphoid organs, where they lose the antigen-processing activity and mature to become potent immunostimulatory cells [10]. The induction of DC maturation is critical for the induction of antigen-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Fully mature DCs show a high surface expression of MHC class II and costimulatory molecules (CD40, CD80, and CD86) but a decreased capacity to internalize antigens [10, 11]. Up-regulation of CD83, a specific marker for DC maturation, also occurs [12]. Various stimuli, such as proinflammatory cytokines (e.g., TNF- and IL-1), CD40 ligation, bacterial products (e.g., LPS and unmethylated DNA CpG motif), and contact sensitizers, can induce DC maturation in vivo and in vitro [13, 14]. Several reports have already indicated that the nuclear transcription factor NF- B also plays an important role in DC maturation [15]. Another intracellular component involved in DC maturation, the three major MAPK signaling pathways in mammals, including the p38 MAPK, ERK, and JNK, are activated in DCs on maturation induced by LPS or TNF- [16]. The exact effects of LZ-8 on human DCs are yet to be defined. In the present study, we first used the yeast Pichia pastoris protein expression systems for producing the immunomodulatory protein LZ-8. Then the rlz-8 protein derived from yeast was treated with human DCs. We also examined the molecular mechanisms of rlz-8 on the activation and maturation of human monocyte-derived DCs. We also investigated its in vivo effect on antigen-specific IgG2a/IgG1 antibody production and ex vivo effect on Th1/Th2 cytokine production by cultured splenocytes derived from OVA-immunized BALB/c mice. MATERIALS AND METHODS Reagents Escherichia coli LPS (L8274, E. coli) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Isotopes were obtained from Amersham Corp. (Piscataway, NJ, USA). Neutralization antibodies (without sodium azide) against TLR1, TLR2, TLR3, and TLR4 were purchased from ebioscience (San Diego, CA, USA), and helenalin, SB203580, PD98059, and JNK inhibitor II were purchased from Calbiochem (Germany). Treatment of immature DCs with these inhibitors (helenalin, SB203580, PD98059, and JNK inhibitor II) before stimulation was performed for 60 min. These inhibitors were dissolved in DMSO, where a 0.1% (v/v) concentration of DMSO was used as a negative control whenever indicated. Plasmid construction and P. pastoris transformation The total DNA of G. lucidum RSH RZ was extracted as described by Al-Samarrai and Schmid [17]. The full length of the lz-8 gene was amplified from chromosomal DNA of G. lucidum by PCR using forward primer 5 - GAATTCATGTCCGACACTGCC-3 and reverse primer 5 -TCTAGATAGT- TCCACTGGGCG-3, and EcoRI and XbaI restriction sites (underlined) were designed for flanking the PCR product at the 5 - and 3 -terminus, respectively. The PCR fragment was cloned into the EcoRI/XbaI site of ppicz A (Invitrogen, Carlsbad, CA, USA) to produce the expression plasmid ppicz A:lz-8. The LZ-8 expression plasmid was transformed into P. pastoris KM71 (his4, aox1::arg4, arg4, Mut s, Invitrogen) according to the manufacturer s instructions. Media and culture conditions for rlz-8 expression The P. pastoris transformant was cultured in a 500-ml Hinton flask containing 100 ml BMGY, supplemented with 1% (v/v) glycerol as a carbon source and 100 g/ml zoecine as a selection pressure. Cells were grown at 30 C and shaken at 250 rpm until an OD 600 value of 20 had been reached, and the cells were then harvested by centrifugation at 3000 g and 4 C for 5 min. They were washed subsequently with potassium phosphate buffer (100 mm, ph 6.0). The pellet was then resuspended in 100 ml BMMY and induced by adding 0.5% (v/v) methanol every 24 h. BMGY and BMMY media were also prepared according to the manufacturer s instructions (Invitrogen). After 48 h of induction, the supernatant was collected by centrifugation at 12,000 g and 4 C for 20 min. rlz-8 protein was purified by a nickel affinity column NiTA-agarose (Qiagen, Valencia, CA, USA) and eluted by a gradient of mm imidazol. Endotoxin levels of rlz-8 were assayed by Limulus amoebocyte lysate assays ( 1.0 EU/ g). Human DC generation Human DCs were generated from PBMC, as described previously [18 20], with some modification. Briefly, PBMCs were obtained from healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). CD14 cells were purified by positive selection using anti-cd14 microbeads in conjunction with the MiniMACS system (Miltenyi Biotech, Auburn, CA, USA). The CD14 cells were cultured at cells/1 ml crpmi in 24- well plates (Costar, Cambridge, MA, USA) with GM-CSF (800 U/ml) and IL-4 (500 U/ml). Fresh medium containing GM-CSF and IL-4 was added every 2 3 days. Human monocyte-derived DCs were used routinely at Day 6 of culture. Determination of cytokine levels The IL-12 p40, IL-10, IL-23, IL-8, IL-5, and IFN- in the culture supernatant from DCs or T cells were assayed with an ELISA kit (R&D Systems, Minneapolis, MN, USA), as per the manufacturer s instructions. Their detection sensitivities were 32, 10, 8, 8, 12, and 8 pg/ml, respectively. Flow cytometric analysis DCs were harvested and washed with cold buffer (PBS containing 2% FCS and 0.1% sodium azide). Cells were then incubated in cold buffer. Subsequent stainings with mab or isotype-matched controls were performed for 30 min on ice. The stained cells were then washed twice and resuspended in cold buffer and analyzed with a FACSort cell analyzer (Becton Dickinson, San Jose, CA, USA). More than cells were analyzed for each sample, and the results were processed using CellQuest software (Becton Dickinson). FITC-labeled dextran uptake Cultured DCs were washed twice and resuspended in 1 ml crpmi The cells were then incubated with FITC-labeled dextran (0.2 mg/ml) at 4 C or 37 C for 1 h. Finally, the cells were washed thrice with cold buffer and analyzed with a FACSort cell analyzer, as described above. Allogeneic MLR PBMCs were obtained as described above, and naïve CD4 T cells were purified from PBMC using magnetic beads (Miltenyi Biotec, Auburn, CA, USA). The allogeneic, naïve CD4 T cells obtained were distributed at cells/well and incubated for 5 days in the presence of graded numbers of irradiated DCs (3000 rad, 137 Cs source). Tritiated thymidine (1 Ci/ well, New England Nuclear, Boston, MA, USA) incorporation for 16 h was determined with a liquid counter. Neutralization experiments Human DCs were preincubated for 1 h with 20 g/ml antibody solution of TLR1, TLR2, TLR3, and TLR4. rlz-8 was then added for 16 h. The cell- 878 Journal of Leukocyte Biology Volume 86, October

3 Lin et al. LZ-8 induces dendritic cell activation culture supernatants were collected and were analyzed for IL-12 p40 and IL-10 by ELISA. Transfection of TLR in HEK293 cells The puno-htlr4, pduo-md2/tlr4, and puno empty plasmids were obtained from InvivoGen (San Diego, CA, USA). The HEK293 cells (American Type Culture Colleciton, Manassas, VA, USA) were plated into six-well tissue-culture plates and maintained in DMEM supplemented with 10% FCS. Cells were transfected using the Lipofectamine TM 2000 transfection protocol (Life Technologies, Carlsbad, CA, USA) with 4.0 g puno, puno-htlr4, pduo-md2/tlr4, and 6 l Lipofectamine. Transfected cells were selected in medium containing 10 g/ml blasticidin S-supplemented medium. Transfected cells were cultured in normal medium for h and then were stimulated with medium alone, rlz-8 (5 g/ml), or LPS (50 ng/ml) for 16 h. IL-8 protein in the culture supernatant was measured by ELISA. IKK activity assay The kinase activity assay was performed as described by Spiecker et al. [21] with some modifications. Whole cell extract was lysed with Gold lysis buffer for 30 min at 4 C. The cell lysate was clarified by centrifugation at 12,000 g for 10 min at 4 C. Equal amounts of total cellular protein (100 g) were immunoprecipitated with IKK1- and IKK2-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and protein A/G-PLUS agarose for 12 h at 4 C. Kinase assay was carried out in 45 l kinase buffer (40 mm Tris-NaOH, ph 7.5, 500 mm NaCl, 0.1% NP-40, 6 mm EDTA, 6 mm EGTA, 10 mm -glycerophosphate, 10 mm NaF, 10 mm p-nitrophenylphosphate, 300 M sodium orthovanadate, 1 mm benzamidine, 2 M PMSF, 10 g/ml aprotinin, 1 g/ml leupeptin, and 1 mm DTT) containing 5 M cold ATP, 10 Ci [ - 32 P] ATP (5000 Ci/mmol, Amersham Corp.), and 1 g GST- I B fusion protein (Santa Cruz Biotechnology) as substrate and incubated for 20 min at 25 C. Each sample was mixed with 8 l 5 Laemmli s loading buffer to stop the reaction, heated for 10 min at 100 C, and subjected to 10% SDS-PAGE. The gels were dried, visualized by autoradiography, and quantified by densitometry (IS-1000 Digital Imaging System). Western blotting Total cellular extract was prepared using Gold lysis buffer. Total protein was separated on 10% SDS-polyacrylamide minigels and transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), which was incubated overnight at 4 C with 10% BSA in PBS to block nonspecific Igs and then incubated with anti-p-i B polyclonal, anti- -tubulin mab (Santa Cruz Biotechnology), anti-p-p38, anti-p-p42/44, anti-p-p46/ 54, and anti-total p38 polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA). Preparation of nuclear extracts and EMSA Nuclear and cytoplasmic extracts were prepared as described previously [22]. Each 5 g nuclear extract was mixed with the labeled, double-stranded NF- B oligonucleotide, 5 -AGTTGAGGGGACTTTCCCAGGC-3, and incubated at room temperature for 20 min. The incubation mixture included 1 g polydeoxyinosinic:polydeoxycytidylic acid in a binding buffer (25 mm HEPES, ph 7.9, 0.5 mm EDTA, 0.5 mm DTT, 1% NP-40, 5% glycerol, and 50 mm NaCl). The DNA protein complex was electrophoresed on 4.5% nondenaturing polyacrylamide gels in M Tris, M borate, M EDTA buffer. A double-stranded, mutated oligonucleotide, 5 -AGTTGAGGCGACTTTCCCAGGC-3, was used to examine the specificity of the binding of NF- B to DNA. The specificity of binding was also examined by comparison with the unlabeled oligonucleotide. Mice and immunizations Female BALB/c mice were obtained from the Animal Center of the College of Medicine, National Taiwan University (R.O.C.). Mice receiving only antigen were immunized by i.p. injections with 0.2 ml solution containing 50 g OVA (Sigma Chemical Co.) in saline. The group receiving experimental adjuvant was immunized with 0.2 ml solution containing 500 g rlz-8 admixed with 50 g OVA in saline. The mice given i.p. injection of 1 PBS in each immunization were regarded as the negative control group. Animals were immunized on Days 0, 14, 28, and 42. Blood was collected by retro-orbital puncture at various time-points after immunization. OVA-specific antibody assay Sera anti-ova IgG1 and IgG2a antibody titers were determined by ELISA. Briefly, 96-well flat-bottom plates were coated with 10 ìg/ml OVA. After overnight incubation at 4 C, plates were washed and blocked with 2% BSA in PBS for 2 h at 37 C. Serum samples were diluted and added to each well overnight at 4 C. Then the plates were washed, and biotin-conjugated anti-mouse IgG1 (1:5000, PharMingen, San Diego, CA, USA) or IgG2a (1: 1000, PharMingen) was added for 1 h at 37 C. Streptavidin-conjugated HRP (1:10,000) was added for an additional 2 h at room temperature. Finally, the reaction was developed by H 2 O 2 and tetramethylbenzidine, followed by 50 l/well H 2 SO 4 stop solution. Absorbance at 450 nm was measured using a microplate reader (Spectra Max M5, Molecular Devices, Sunnyvale, CA, USA). The results were expressed in EU: EU (A sample A blank )/(A positive A bl ank ). Determination of cytokine levels in spenocyte culture To measure the levels of cytokines, splenocytes ( /well) of immunized mice treated with or without rlz-8 were cultured in RPMI-1640 medium supplemented with 2% tissue culture medium in the presence of OVA (10 g/ml) in 24-well microtiter plates at 37 C for 48 h. The culture supernatants were collected and centrifuged at 400 g at 4 C. The cell-free supernatants were stored at 70 C until they were used for the cytokine assay. The IFN-, IL-2, and IL-5 in the culture supernatants were assayed with an ELISA kit (R&D Systems) according to the manufacturer s instructions. Statistical analysis The Student s t-test was used to analyze the results, and a P value of less than 0.05 was considered to be statistically significant. RESULTS rlz-8 induces IL-12 p40, IL-10, and IL-23 production and maturation of human monocyte-derived DC LPS has been described as an inducer of DC activation and maturation. We use LPS as a positive control in this study. To determine whether rlz-8 can affect the cytokine production in human monocyte-derived DC, we compared cytokine concentrations in the supernatants of DC cultured with different doses of rlz-8. Our data demonstrated that LPS and rlz-8 can enhance the production of IL-12 p40, IL-10, and IL-23 (Fig. 1A). When human DCs were treated with 10 g/ml rlz-8 for 24 and 48 h, we found that rlz-8 enhanced the production of IL-12 p40 and IL-10 significantly (Fig. 1B). It was clear that the stimulatory effect of rlz-8 on IL-12 p40, IL-10, and IL-23 production was dose- and time-dependent in manner. However, the level of IL-23 is low in rlz-8-stimulated DC compared with LPS-stimulated DC. The negative protein from mock-transformed P. pastoris cannot induce IL-12 p40 and IL-10 produc- Volume 86, October 2009 Journal of Leukocyte Biology 879

4 Figure 1. rlz-8 effects on DC maturation. (A) Human DCs were cultured for 24 h in the presence of various concentrations of rlz-8 or LPS (100 ng/ml). At the end of the incubation time, the culture medium was collected for cytokine assay by ELISA. (B) Human DCs were incubated with rlz-8 (10 g/ml) for 24 and 48 h. At the end of the incubation time, IL-12 p40, IL-10, and IL-23 production was analyzed subsequently by ELISA. Each data represent the mean se for three determinations. Statistical analysis concerns unstimulated versus stimulated DC. *, P (C) Human DCs were treated with rlz-8 (10 g/ml), LPS (100 ng/ml), or medium alone for 24 h, and surface markers were analyzed by flow cytometry (dotted line, isotype control; solid line, specific mab). The values shown in the flow cytometry profiles are the MFI indexes. This experiment was repeated three times with similar results. (D) The flow cytometry data are expressed as MFI se. *,P tion (data not shown). Trypan blue exclusion analysis, done at the end of each experiment to test for cell viability, showed that at the concentrations used, none of the compounds had a toxic effect on the cells (data not shown). Next, to investigate whether rlz-8 can also modulate the development of human DCs in vitro, we compared the phenotype of human DCs treated with medium alone, LPS, or rlz-8 for 24 h. The data demonstrated that rlz-8 and LPS increased the presentation of CD80, CD86, CD83, and MHC class II molecules on the cell surface of human DCs (Fig. 1, C and D). Proteinase K-treated rlz-8 abrogates IL-12 p40 and IL-10 production in human DCs To rule out the possibility of endotoxin contamination in rlz-8, we used proteinase K (1 mg/ml) to degrade rlz-8. Proteinase K exhibits broad substrate specificity and degrades many proteins [23]. The results demonstrated clearly that rlz-8 induced IL-12 p40 and IL-10 production effectively in human DCs, but proteinase K-treated rlz-8 did not induce any production of IL-12 p40 and IL-10, and the levels of IL-12 p40 and IL-10 were similar to the control group (Fig. 2A). In Figure 2B, the LPS-positive control was also treated with proteinase K. The data demonstrated that proteinase K-treated LPS could not affect the induction of IL-12 p40 and IL-10 production in human DCs. Thus, the results indicated that it was unlikely that rlz-8 was contaminated with endotoxin. rlz-8 down-regulation of endocytotic activity of human DCs Immature DCs capture and process antigens via their high endocytic capacity. They lose their endocytic/processing capacity of antigens and mature into potent immunostimulatory APCs during differentiation [8]. The uptake of FITC-dextran is known to be maximal in the immature monocyte-derived DC and occurs by a combination of macropinocytosis and binding to the mannose receptor. LPS has been described as an inducer of DC activation and maturation [24]. Therefore, we used LPS as a positive control in this study. Previous studies have shown that the endocytic capacity of DCs is suppressed by LPS during their maturation process. Thus, we tested whether rlz Journal of Leukocyte Biology Volume 86, October

5 Lin et al. LZ-8 induces dendritic cell activation Figure 2. Proteinase K-treated rlz-8 abrogated IL-12 p40 and IL-10 production in DCs. (A) Human DCs were incubated with medium alone or rlz-8 (10 g/ml), or proteinase K (1 mg/ml)-treated rlz-8 for 24 h. (B) Human DCs were incubated with medium alone, LPS (100 ng/ml), or proteinase K (1 mg/ml)-treated LPS for 24 h. At the end of the incubation time, IL-12 p40 and IL-10 production was analyzed subsequently by ELISA. Each data represent the mean se for three determinations. Statistical analysis concerns rlz-8-stimulated DC versus proteinase K-treated rlz-8-stimulated DC or LPS-stimulated DC versus proteinase K-treated LPS-stimulated DC. *, P This experiment was repeated three times with similar results. rlz-8 induces IL-12 p40 and IL-10 synthesis through TLR4 TLRs have been shown to be involved in the antifungal defense mechanism in Drosophila and antibacterial defense in humans. To examine whether these receptors were involved in the rlz-8-mediated signal transductions in human DCs, neutralization experiments were performed. Cell-surface TLR1, TLR2, TLR3, and TLR4 receptors were blocked by neutralizing concentrations of their respective antibodies before DCs were treated with rlz-8. We demonstrated that the addition of an anti-tlr4 mab to human DCs blocked rlz-8 (10 g/ml)-induced IL-12 p40 and IL-10 production significantly by 64% and 77%, respectively, but the addition of anti-tlr1, -TLR2, and -TLR3 mab failed to inhibit rlz-8-induced IL-12 p40 and IL-10 production (Fig. 5A). To confirm further that rlz-8 uses TLR4, we tested whether rlz-8 could activate HEK293 cells expressing functional TLR4 by assessing IL-8 production. The up-regulation of IL-8 gene transcription is known to involve the activation of the NF- B signaling pathway. The capacity of rlz-8 or LPS to induce IL-8 production by transfected HEK293 cells was affected the uptake of FITC-labeled dextran by human DCs. In our study, we demonstrated a reduction in FITC-dextran uptake when human DCs were matured with rlz-8 (Fig. 3). Enhancement of T cell activation by LZ-8-treated human DCs Mature DCs have the capacity to induce proliferation in allogenic T cells at a much higher level than immature DCs [10]. In human DCs, we found that rlz-8 up-regulated cell-surface markers, increased IL-12 production, and induced the activation of NF- B. To test whether this maturation is sufficient to promote activation of naive T cells, we first investigated the impact of rlz-8 treatment of human DCs on their capacity to present antigen and subsequently stimulate allogeneic T cells. Human DCs were treated and untreated with rlz-8 as stimulator APCs. These cells were then used to activate allogenic, naive T cells. As shown in Figure 4A, the proliferative response of allogeneic T cells was induced effectively by rlz-8 treatment of DCs. The results presented in Figure 4B showed that rlz-8-treated DCs enhanced T cell activation as evidenced by the secretion of IFN- in the culture supernatant. We also demonstrated that rlz-8-treated DCs enhanced T cell secretion of IL-10 and IL-5 in the supernatant (Fig. 4B). In the rlz-8-treated DCs or the LPStreated DCs, both DCs could not induce T cell secretion of IL-4 cytokine (data not shown). Figure 3. rlz-8 on the endocytotic capacities of human DCs. At Day 6, immature DCs were stimulated with medium alone, LPS (100 ng/ml), or rlz-8 (10 g/ml) for 24 h, and cells were then incubated with FITC-dextran for 1 h at 4 C(dotted lines) or 37 C (solid lines). This experiment was repeated three times with similar results. Volume 86, October 2009 Journal of Leukocyte Biology 881

6 Thus, these results suggest that TLR4 is involved in rlz-8- regulated IL-12 p40 and IL-10 expression. rlz-8 induces IKK activity, phosphorylation of I B, and NF- B activation in human DCs As the activation of IKK activity is necessary for I B phosphorylation, the effect of rlz-8 on IKK activity was studied. Human DCs were treated with rlz-8 (10 g/ml) for the indicated periods of time. To measure IKK1 activity directly in human DCs, IKK1 and IKK2 proteins were immunoprecipitated from cell extracts, and the kinase activity in the immunocomplex was assayed using rgst-i B (1 317) as a substrate. Figure 6A illustrates the relative effect on IKK activity. After stimulation with rlz-8, the GST-I B fusion protein was strongly phosphorylated at 45 min, indicating the stimulation of IKK activity in human DCs. NF- B is one molecular family whose activation is associated with DC maturation. NF- B normally binds to I B, which impedes NF- B nuclear translocation from the cytoplasm to the nucleus. Once cells are exposed to inflammatory stimuli, including LPS and TNF-, I B is phosphorylated, leading to I B degradation and nuclear translocation of NF- B. We thus studied whether rlz-8 had any affect on I B phosphorylation. The cytoplasmic levels of P-I B protein were examined by Western blot analysis. After 120 min from the activation of human DCs with rlz-8, the cytosolic I B protein was phosphorylated significantly (Fig. 6B). DC maturation derived by LPS has been clearly associated with NF- B activation. To assess the action of rlz-8 in DC gene expression and function by a similar intracellular signaling pathway, we monitored its ability to activate NF- B translocation into the nucleus. DCs were cultured in the presence of LPS or rlz-8 for 2 h, and nuclear extracts were analyzed for NF- B binding by the EMSA. As shown in Figure 6C, rlz-8 was able to induce NF- B translocation and activation. Identical results were obtained after treatment of DCs with LPS. The binding of NF- B was specific and could be blocked by unlabeled, competing NF- B oligonucleotide. Figure 4. rlz-8-treated DCs enhance T cells response. (A) Immature DCs were stimulated with medium alone, rlz-8 (10 g/ml), or LPS (100 ng/ml) for 24 h. Allogeneic T cell proliferation was measured after 5 days of coculture with irradiated, untreated DCs, rlz-8-treated DCs, or LPS-treated DCs. Supernatants were analyzed for (B) IFN-, IL-5, and IL-10 produced by activated T cells after 2 days of culture. These data are means sem of triplicates and representative of three independent experiments. *, P tested (Fig. 5B). TLR4 or TLR4/MD2-transfected HEK293 cells produced IL-8 in response to rlz-8 or LPS. However, medium alone did not induce significant IL-8 production from HEK293 cells transfected with TLR4 or TLR4/MD2. Induced phosphorylation of members of the three MAPK families in rlz-8-treated human DCs MAPK is a serine and threonine protein kinase whose activities are up-regulated through tyrosine and threonine residue phosphorylation by its upstream regulators [25]. This experiment focused on p38 MAPK, p42/44 ERK, and p46/54 JNK to further characterize the MAPK activation pathways involved in rlz-8 signaling. Human DCs were stimulated with rlz-8 at the indicated periods of time, and the level of MAPK phosphorylations was assessed by Western blotting with respective antityrosine-phosphorylated MAPK mab. Total p38 mab was used for an internal control. Results presented in Figure 7 show that rlz-8 induced the phosphorylation of all MAPKs tested, especially in inducing a higher p38 MAPK and p42/44 ERK phosphorylation at 120 min and inducing a higher p46/54 JNK phosphorylation at 45 min and 60 min. The total amount of p38 was unchanged following stimulation. 882 Journal of Leukocyte Biology Volume 86, October

7 Lin et al. LZ-8 induces dendritic cell activation Figure 5. rlz-8-induced DC maturation is mediated by TLR4. (A) Neutralization with TLR4 mab inhibits the synthesis of IL-12 p40 and IL-10 in rlz-8-treated human DCs, which were preincubated with 20 g/ml anti-tlr1, -TLR2, -TLR3, and -TLR4 antibodies separately for 1 h. DCs were then challenged with rlz-8 (10 g/ml) for 15 h. The cell culture supernatants were collected for IL-12 p40 and IL-10 analysis. A significant difference between DCs treated with antibodies and no antibodies is indicated by *, P (B) HEK293 cells were transfected with control plasmids (Cont), TLR4, or TLR4 MD2. The transfected cells were stimulated with medium alone, rlz-8 (5 g/ml), or LPS (50 ng/ml) for 24 h, and IL-8 protein in the supernatants was measured by ELISA. Data of three experiments are represented as mean se. *,P Inhibition of NF- B and MAPKs prevents the maturation changes induced by rlz-8 rlz-8-treated DCs induced IL-12 p40 and IL-10 production during maturation (Fig. 1, A and B). We investigated whether the rlz-8-induced secretion of IL-12 p40 and IL-10 was affected by the inhibitors of NF- B, p38 MAPK, p42/44 ERK, and p46/54 JNK. Immature human DCs were pretreated with helenalin (a specific blocker of NF- B), SB (a specific blocker of p38 MAPK), PD98059 (an inhibitor of the ERK pathway), or JNK inhibitor II (an inhibitor of the JNK pathway) for 1 h and subsequently stimulated with rlz-8 for 24 h. The production of IL-12 p40 and IL-10 was quantified by means of ELISA. rlz-8 induced significant production of IL-12 p40 and IL-10; the IL-12 p40 cytokine productions were abrogated significantly by helenalin, SB203580, PD98059, and JNK inhibitor II; and the IL-10 was inhibited completed by helenalin, PD98059, and JNK inhibitor II (Fig. 8A). In contrast, SB only had an effect on down-regulated IL-12 p40 but no effect on the inhibition of IL-10 production induced by rlz-8. To elucidate further the role of NF- B, p38 MAPK, ERK, and JNK signaling pathways in the rlz-8-induced expression of costimulatory and antigen-presenting molecules, NF- B inhibitor helenalin, p38 MAPK inhibitor SB203580, ERK pathway inhibitor PD98059, and JNK pathway inhibitor JNK inhibitor II, in the expression of costimulatory and adhesion molecules as well as HLA-DR, were investigated. Blocking the NF- B pathway with helenalin inhibited the rlz-8-induced up-regulation of CD80, CD86, CD83, and HLA-DR significantly (Fig. 8B). Blocking the p38 MAPK, ERK, and JNK pathway with SB203580, PD98059, and JNK inhibitor II, respectively, also had an effect on CD80, CD86, CD83, and HLA-DR expression. These results show that certain features of human monocytederived DC maturation are regulated by signaling via NF- B and MAPK and imply that different aspects of the maturation process induced by rlz-8 may be regulated by distinct signal transduction pathways. Effect of rlz-8 on serum anti-ova antibody levels To confirm the microarray data, we studied the effect of rlz-8 on antigen-specific IgG1 and IgG2a in OVA-immunized BALB/c mice. We obtained serum from OVA immunized 42 and 56 days after the first immunization. In rlz-8 admixed with OVAimmunized mice, antigen-specific IgG2a production was increased significantly at the time-point (P , Day 42; P , Day 56) examined after immunization as compared with mice immunized with OVA alone (Fig. 9A). The anti-ova IgG1 antibody levels showed significant differences at Day 42 (P ) but no significant differences at Day 56 (P ) among the OVA-immunized mice (Fig. 9B). Regulatory effect of rlz-8 on the balance of Th1/ Th2 cell responses in OVA-immunized mice It has been reported that cytokines play an important role in the antibody response. Therefore, we examined the regulatory effect of rlz-8 on Th1/Th2 cell responses in OVA-immunized mice, which were also immunized with OVA plus rlz-8 on Days 0, 14, 28, and 42. IFN-, IL-2 (Th1 cytokine), and IL-5 (Th2 cytokine) production in splenocytes stimulated with OVA was assayed (Fig. 10). The production of IFN- and IL-2 increased significantly in rlz-8 admixed with OVA-immunized mice compared with OVA-immunized mice (P , IFN- ; P , IL-2). The production of IL-5 showed no significant differences in rlz-8 admixed with OVA-immunized mice compared with OVA-immunized mice (P ). These results indicate that rlz-8 changed the balance of Th1/Th2 cell immune responses from Th2-dominant to Th1-dominant in OVAimmunized mice. DISCUSSION Polysaccharides and triterpenes have been investigated most thoroughly from G. lucidum and related species [26]. However, sterols, nucleosides, alkaloids, and proteins have also been de- Volume 86, October 2009 Journal of Leukocyte Biology 883

8 Figure 6. rlz-8 induced IKK activity, I B phosphorylation, and NF- B activation in human DCs. (A) Human DCs were treated with rlz-8 (10 g/ml) for the indicated time periods and the total cell lysates collected for IKK activity assay. Immunoprecipitated IKK was incubated with [ - 32 P] ATP and GST-I B fusion protein as substrates. The kinase activity assay was performed as described in Materials and Methods, and 32 P-labeled GST-I B values are shown. (B) Human monocyte-derived DCs were treated with rlz-8 (10 g/ml) for the indicated time periods. Cytosolic fractions were prepared and analyzed for the phosphorylation level of I B by Western blotting. The lower panel shows the blot probed for -tubulin to demonstrate equal loading of samples. The units represent the relative fold of P-I B/ -tubulin. (C) Human DCs were treated with LPS (100 ng/ml) or rlz-8 (10 g/ml) for 2horremained unstimulated. Nuclear fractions were prepared and analyzed for NF- B binding activity by EMSA. To assess the specificity of the binding, 100-fold excess of cold NF- B probe or mutant probe was added to the rlz-8 group. Band intensities were quantified by densitometry. The arbitrary units represent the relative amounts of the radioactivity present in the respective band. This experiment was repeated three times with similar results. scribed. Bioactive proteins have been isolated and characterized by chromatographic/electrophoretic techniques. For example, a new immunomodulatory protein, known as LZ-8, was isolated from the mycelia of G. lucidum [5]. Kino et al. [5] reported that LZ-8 has mitogenic activity in vitro and immunomodulating activity in vivo. They also examined the hemagglutination activity of LZ-8 with sheep and human RBCs, revealing the ability to aggregate only SRBCs and not human RBCs of any type. The lack of hemagglutination activity toward human RBCs may prove advantageous if LZ-8 is developed for therapeutic use in the future. However, the traditional method to generate a specific immunomodulatory protein from G. lucidum is limited as a result of the problem of other unnecessary molecule contamination and expensive but low-yield production. Recombinant technologies can be used to prevent these problems. Therefore, we developed a large-scale P. pastoris protein expression system for rlz-8 expression. DCs are the crucial cell type at the interface between innate and adaptive immune responses, and they are capable of activating naïve T cells [27]. This process is regulated by various extracellular stimuli, including cytokines, bacterial products, and membrane-bound ligands [28, 29]. Recently, Cao and Lin [30] showed that G. lucidum polysaccharides could promote the maturation and function of murine DCs. These results are similar to our findings in human DCs [31]. We demonstrated that PS-G could effectively and rapidly induce the significant activation and maturation of human DCs by the NF- B and p38 MAPK pathways. However, little is known about the molec- 884 Journal of Leukocyte Biology Volume 86, October

9 Lin et al. LZ-8 induces dendritic cell activation Figure 7. rlz-8 induces the phosphorylation of p38 MAPK, p42/44 ERK, and p46/52 JNK kinase. Human monocyte-derived DCs were treated with rlz-8 (10 g/ml) for the indicated time periods, then the cell lysates were collected, and the level of MAPK phosphorylations was assessed by Western blotting with respective antityrosine-phosphorylated MAPK mab and total p38 mab for internal control. ular mechanisms responsible for the regulation of DCs in their activation and maturation states by LZ-8. In this study, we are the first to demonstrate the rlz-8-induced morphological, phenotypical, and functional changes in human monocytederived DCs. rlz-8 promoted the maturation of DCs, and mature DCs demonstrated characteristic morphology with enlarged size and numerous cytoplasmic processes that gave rise to a stellate appearance (data not shown). Fully mature DC show a high surface expression of MHC class II, co-stimulatory molecules (CD80 and CD86), and up-regulation of CD83, a specific marker for DC maturation, also occurs. rlz-8-treated DCs can induce MHC class II, CD80, CD86, and CD83 expression, as well as the enhanced production IL-12. In T lymphocyte growth and differentiation, IL-12 played an important role. The CD83 marker for mature human DCs was also increased. IL-12 and IL-23, two heterodimeric cytokines produced by APCs, are composed of a specific polypeptide (namely, p35 for IL-12 and p19 for IL-23) disulfide linked to a common p40 chain to form the biologically active molecules. In immune responses, IL-12 plays a central role as a link between the innate and adaptive immune systems [32]. Thus, IL-12 induces and promotes NK and T cells to generate IFN- and lytic activity. In addition, IL-12 polarizes the immune system toward a primary Th1 cell response. IL-23 is produced by different cell types, including macrophages and DCs. IL-23 is not as efficient as IL-12 in the induction of IFN- production and in the polarization of T cells to the Th1 pattern. On the other hand, IL-23 is more effective than IL-12 in the induction of memory T cell proliferation. However, in addition to its role in memory T cell responses, IL-23 drives the development of a novel T cell subset characterized by the production of IL-17 (Th17), which plays a central role in mediating chronic inflammatory responses [33]. In this study, we found that rlz-8 can induce IL-12 p40, IL-10, and IL-23 production in human DCs. However, the level of IL-23 is low in rlz-8-stimulated DC. Therefore, we suggested that IL-12 p40 and IL-10 play the major role in control of rlz-8-mediated DC maturation and activation. The biological function of IL-12 p40 is still debated. IL-12 p40 is a natural antagonist, which inhibits IL-12- and IL- 23-mediated biological activity by blocking the binding of IL- 12/23 to their receptors. Recently, IL-12 p40 acts as a chemoattractant for macrophages and promotes the migration of bacterially stimulated DCs [34]. It was also shown to have immune-enhancing activity through the activation of macrophages or DCs [35]. Recently, Jana and Pahan [36] found that p40 2 induced the expression of lymphotoxin- in microglia and macrophages via IL-12R 1, the binding receptor of the high-affinity IL-12R complex. Therefore, they suggested that p40 2 is biologically active and suggest that p40 2 may be considered as a separate cytokine with biological functions distinct from IL-12 p70 [36]. IL-10 is a pleiotropic cytokine produced by DCs, T cells, and macrophages and possesses anti-inflammatory and immunosuppressive properties [37]. The regulatory circuit involved IL-12 and IL-10 has been proposed and described in an autoimmune disease model [38, 39]. Segal et al. [39] demonstrate that IL-12 is essential for the generation of the autoreactive Th1 cells that induce EAE. The disease-promoting effects of IL-12 are antagonized by IL-10, produced by an antigen-nonspecific CD4 T cell. However, it is not required for the development of EAE, as demonstrated by recent studies in IL-12p35 / mice [40, 41]. Using IL-23p19 / mice, Cua et al. [42] found that IL-23, but not IL-12, plays a critical role in the induction of EAE. In this study, we suggest that when IL-12 p40 is overexpressed in rlz-8-treated human DCs, IL-10 can act as a feedback regulatory role. At a functional level, rlz-8-induced DC maturation resulted in an enhanced allostimmulatory activity, as demonstrated by enhanced proliferative responses of naïve, allogeneic T cells. In addition, a higher level of IFN- and lower levels of IL-10 and IL-5 were induced in MLR by rlz-8-treated human DCs. Therefore, our experimental data show that rlz-8 modulates the function of human DCs in a manner that results in a preferential induction of Th1-dominant adaptive immune responses. However, under some conditions, rlz-8-treated DCs can promote the differentiation of naïve T cells for the secretion of IL-10. TLRs have been identified in humans as an important component of innate immunity against microbial pathogens [43]. The role of TLR4 in Gram-negative bacterial LPS-mediated signaling has been studied extensively; however, the role of TLR in response to rlz-8 is less studied and unclear in human DCs. Neutralization experiments with antibodies blocking TLR1, TLR2, TLR3, and TLR4 demonstrated further that TLR4 plays a critical role in the signal transduction cascade induced by rlz-8. We also used TLR4, or TLR4/MD2-transfected HEK293 cells produced IL-8 in response to rlz-8 to confirm. The result also demonstrated that MD2 is required for rlz-8 or LPS to activate IL-8 generation. Recent reports show that LPS and TNF-, two potent DC maturation factors, induced the NF- B activation and phosphorylation of p38, ERK1/2, and p46/54 JNK in monocyte-derived DCs [44]. Our results demonstrated that rlz-8 activated NF- B and all three MAPK pathways during DC maturation. Recent reports show that NF- B is responsi- Volume 86, October 2009 Journal of Leukocyte Biology 885

10 Figure 8. The effect of inhibiting the NF- B, p38 MAPK, ERK1/2, or JNK pathways on the rlz-8-induced up-regulation of IL-12 p40 and IL-10 production and CD80, CD86, CD83, and HLA-DR expression in human monocyte-derived DCs, which were pretreated with 0.1% DMSO, 10 M helenalin (a specific blocker of NF- B), 20 M SB (a specific blocker of p38 MAPK), 50 M PD98059 (an inhibitor of the ERK1/2 pathway), or 20 M JNK inhibitor II (an inhibitor of the JNK pathway) for 1 h and then incubated with 10 g/ml rlz-8 for 24 h. (A) At the end of the incubation time, the supernatant was collected for IL-12 p 40 and IL-10 production by ELISA. A significant difference between DC treated with rlz-8 alone and pretreated with inhibitors is indicated by *, P (B) The cell-surface expression of CD80, CD86, CD83, and HLA-DR was then measured using the flow cytometry (dotted line, isotype control; solid line, specific mab). The values shown in the flow cytometry profiles are the MFI indexes. These results are representative of three independent experiments with similar results. ble for LPS-induced DC maturation in an in vitro murine model [45] and that cytokine-induced maturation of human DCs results in increased NF- B nuclear translocation [46]. Many proinflammatory cytokines displayed NF- B-responsive elements in their promoters, conferring a major role on immune responses [47]. Moreover, the p38 MAPK pathway has been shown to contribute to NF- B-mediated transactivation [48, 49]. Little is known about the signal transduction pathways involved in the maturation of human monocyte-derived DCs by rlz-8. Early phosphorylation of p38 MAPK, ERK1/2, and p46/54 JNK was investigated in rlz-8-treated human monocyte-derived DCs. We demonstrated that the NF- B pathway, the p38 MAPK, the ERK1/2, and the p46/ 54 JNK pathways are all activated when immature human DCs are exposed to rlz-8, suggesting a role of these pathways in the maturation process. The promoters of the hil-12 p40 gene contain B-binding sites [50]. It is likely that NF- B is involved in the IL-12 p40 expression. The availability of specific inhibitory drugs for the NF- B, p38 MAPK, ERK, and JNK pathways prompted us to investigate the respective roles of the NF- B and these MAPKs in DC maturation. In cytokine analysis, pretreatment of helenalin inhibited the IL-12 p40 and IL-10 productions significantly in rlz-8- treated human DCs. PD98059 and JNK inhibitor II were shown to inhibit IL-10 production, although we only observed 50 60% inhibitory effect of these compounds in the up-regulation of IL-12 p40 in the process of DC maturation triggered by rlz-8. In contrast, SB only had an effect on down-regulated IL-12 p40 but no effect on the inhibition of IL-10 production induced by rlz-8. Concerning costimulatory molecules and MHC class II expression, helenalin-pretreated human DCs were able to suppress completely these molecules expression induced by rlz-8. The inhibition of p38 MAPK, ERK1/2, and p46/54 JNK by SB203580, PD98059, and JNK inhibitor II, respectively, before rlz-8 stimulation also had some effect on CD80, CD86, CD83, and MHC class II expression induced during DC maturation. Moreover, the inhibitory effects of these inhibitors were not a result of nonspecific toxicity, as the viability of DCs was not modified by these inhibitors (data not shown). Collectively, these results show that the NF- B and MAPK pathways play the critical roles in the initiation of DC maturation. It appears that the different aspects of 886 Journal of Leukocyte Biology Volume 86, October

11 Lin et al. LZ-8 induces dendritic cell activation responses. The precise mechanism by which rlz-8 induced Th1 responses in vivo might be that the rlz-8 can induce DC activation and maturation and thus, induce Th1-related cytokine production. The signaling pathway elicited by rlz-8 in the production of IL-12 p40 and IL-10 in human DCs is summarized in Figure 11. We demonstrated that binding of rlz-8 to TLR4 could effectively and rapidly induce the significant activation and maturation of human DCs by the NF- B and MAPK pathways. These data suggest that rlz-8 is a good and potential part of the treatment regimen to regulate host immune responses. Lin et al. found that a mutant with deletions at Leu-5, Phe-7, and Leu-9 lost the amphipathic characteristics of the N-terminal domain and the ability to form dimers as well as its immunomodulatory activity. These mu- Figure 9. Serum anti-ova IgG2a (A) and IgG1 (B) responses following i.p. immunization of Balb/c mice with OVA or OVA/rLZ-8. Mice were immunized with OVA (50 g, i.p.) plus rlz-8 (500 g, i.p.) or PBS on Days 0, 14, 28, and 42, and serum samples were collected on Days 0, 42, and 56 after the first immunization. Data represent the mean se, and each group had six mice. DC maturation are regulated by different signal transduction pathways. We investigated further the adjuvant effects of rlz-8 on antigen-specific antibody and cytokine production using BALB/c mice immunized with OVA antigen. This study demonstrates that rlz-8 appears to have marked induction effects on Th1 responses, as treatment of mice with rlz-8 was followed by an increase in Th1 response, including anti-ova IgG2a, IFN-, and IL-2 production. These findings demonstrate that rlz-8 could be used as an adjuvant to induce Th1 immunity in BALB/c mice. Taken together, our data demonstrated that rlz-8 could effectively promote the activation and maturation of immature DC and prefer a Th1 response, suggesting that rlz-8 may possess the potential capacity in regulating immune Figure 10. IFN-, IL-2, and IL-5 production by murine splenocytes after immunization of Balb/c mice with OVA plus rlz-8. Mice were immunized as described in Figure 9. The splenocytes were prepared on Day 56 and were incubated at 37 C with OVA (50 g/ml) for 48 h. The culture supernatants were collected and used to determine IFN-, IL-2, and IL-5 production. The data shown are means se. Volume 86, October 2009 Journal of Leukocyte Biology 887